EP2171444B1 - Procédé d'analyse de globules blancs - Google Patents

Procédé d'analyse de globules blancs Download PDF

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EP2171444B1
EP2171444B1 EP08781438.0A EP08781438A EP2171444B1 EP 2171444 B1 EP2171444 B1 EP 2171444B1 EP 08781438 A EP08781438 A EP 08781438A EP 2171444 B1 EP2171444 B1 EP 2171444B1
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labeled
sample
binding compound
binding
fluorescent dye
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EP2171444A1 (fr
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Edward Goldberg
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Becton Dickinson and Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5052—Cells of the immune system involving B-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70507—C2D
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70589—CD45
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • Cell surface markers permit the identification of cellular phenotypes characteristic of both healthy and disease states, e.g. Maecker et al., J. Clin. Immunol., 20 : 391-399 (2000 ); Rothe et al., Leukemia, 10: 877-895 (1996 ); Reilly et al., J. Clin. Pathol., 54: 508-511 (2001 ).
  • Such markers are typically measured by staining a cell sample with a selection of labeled antibodies specific for different markers, followed by multi-parameter analysis by imaging or by flow cytometry, e.g. Stewart, Immunophenotyping (Wiley-Liss, 2000 ). Further J.
  • EP-A-317156 describes the detection and identification of different lineages and stages of cell development in a sample of hematopoietic cells using two types of monoclonal antibodies that are conjugated with different fluorochromes having distinct emission spectra.
  • D.L. Morris et al., J. Pharmacol. Toxicol. Meth. 37(1):37-46 (1997 ) describes immune-typing rat lymphocyte subsets with the flouresecent antibodies.
  • P. Mikolajczak et al., Drug and Alcohol Dependence 60(3): 303-309 (2000 ) describes the measure of absolute and relative number of lymphocytes in specific subsets (CD3, CD4, CD8, NK, CD45RA) by flow cytometry. J.
  • Multi-parameter analysis using labeled antibodies, or immunophenotyping has been particularly useful for identifying distinct functional and developmental classes of white blood cells, which has important applications in the classification of blood-related diseases, such as leukemias and lymphomas, and in the monitoring the status of HIV infected individuals.
  • the invention provides methods and compositions for analyzing white blood cells.
  • the invention provides a method for enumerating absolute counts of lymphocytes in a blood sample, the method comprising the steps of:
  • the invention provides a method for determining the percentage of T-helper lymphocytes in a blood sample, the method comprising the steps of:
  • the invention provides the use of an kit comprising a fluorescent dye-labeled first binding compound specific for a T lymphocyte specific marker that is CD2 or CD3 and a fluorescent dye-labeled second binding compound specific for CD45RA, wherein said first binding compound and said second binding compound are labeled with the same fluorescent dye, for enumerating absolute counts of lymphocytes in a blood sample according the method of the first aspect above or for determining the percentage of T-helper lymphocytes in a blood sample according to the method of the first aspect above.
  • the invention provides a convenient and effective method for identifying lymphocytes, for either absolute counts or percentage of white blood cell measurements, in a blood sample solely on the basis of surface markers, without the need of light scatter-based measurements to distinguish lymphocytes from other white blood cell types, such as monocytes or granulocytes.
  • the invention is particularly well-suited for use with image-based cell identification devices to provide measurements of percent CD4+ populations in whole blood samples.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques from molecular biology (including recombinant techniques), cell biology, immunoassay technology, microscopy, image analysis, and analytical chemistry, which are within the skill of the art.
  • conventional techniques include labeling of biological cells, immunostaining biological cells, detection of fluorescent signals, image analysis and selection of illumination sources and optical signal detection components.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Robinson et al. (Editors) Current Protocols in Cytometry (John Wiley & Sons, 2007 ); Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, (both from Cold Spring Harbor Laboratory Press ); Owens et al.
  • a cell staining composition comprising a mixture of at least two labeled binding compounds one specific for CD45RA and one specific for a cell surface marker common to T-lymphocytes, preferably a T lymphocyte-specific marker, such as CD2 or CD3.
  • the relative proportions of each binding compounds in the composition may vary widely and depend factors well-known to those with ordinary skill in the art, including the expected number of markers on each cell type, the specificity of the binding compounds for the markers, the stability of the marker-binding compound conjugates and the nature of the label employed.
  • Labeled binding compounds used with the invention are readily available from commercial sources or by assembly using conventional techniques, such as disclosed in the above references.
  • binding compounds used in the invention are labeled with molecules that are capable of generating an optical signal.
  • the optical signal is a fluorescent signal generated by one or more fluorescent dye molecules attached to a binding compound.
  • a method of the invention may be implemented by, or include, the following steps: (a) combining under binding compound-binding conditions a blood sample with at least a first binding compound specific for at least T-lymphocytes and a second binding compound distinct from the first binding compound, which second binding compound is specific for CD45RA molecules, wherein the first and second binding compounds being capable of generating a first optical signal; (b) measuring the first optical signal generated by the first and second binding compound on cells in the sample; and (c) determining lymphocytes of sample by the values of the first optical signal from the first and second binding compound. In accordance with the method, lymphocytes will correspond to cell with high signal values.
  • binding compound-binding conditions means reaction conditions that enable, and preferably maximize, the specific binding of the binding compounds to their respective target molecules. Such conditions include both physical and chemical conditions, such as pH, salt and temperature. Chemical conditions may be controlled by use of buffers, e.g. phosphate buffer, and chelating agents. Preferably, binding compounds are monoclonal antibodies, so that conditions for maximizing specificity are well known to those in the art.
  • Binding compounds are combined with a sample using conventional techniques. Usually, binding compounds are combined under antibody-binding conditions in a reaction vessel, which may be a conventional test tube or a microtiter well, after which it is allowed to incubate for a sufficient time to permit stable complexes to formed between binding compounds and their respective target molecules, if present. Selecting volumes, buffer types and concentrations, and other reaction ingredients is well-known to those of ordinary skill, as exemplified in the above references.
  • a sample analyzed by the method of the invention may be any blood sample that contains lymphocytes, including, for example, whole blood, whole blood that has been processed to remove components, isolated white blood cells and lymphatic fluids.
  • the sample employed is whole blood; that is, blood removed from an organism or individual, patient without processing or treatment prior to analysis.
  • Guidance for making measurements on whole blood samples is provided in the following exemplary references U.S. patent 4,882,284 ; 5,627,037 ; 6,951,727 and 4,727,020 .
  • an optional step may be included for removing or eliminating red blood cells from a sample in order to eliminate or reduce their interference in detecting white blood cells.
  • such elimination is accomplished by lysing red blood cells in the sample.
  • the persistence of erythrocytes in the whole blood sample beyond the staining step may complicate later efforts to measure fluorophore bound specifically to the nucleated cells in the sample.
  • the methods of the present invention may include, for example, after the antibody incubation step and before the flow cytometric acquisition of data, a further step of lysing the erythrocytes in the sample.
  • a number of agents that serve simultaneously to lyse red blood cells and to fix nucleated cells in a whole blood sample, without interfering with binding of antibody to the nucleated cells, are known in the art. These agents are described, inter alia, in U.S. Pat. Nos. 4,654,312 ; 4,902,613 ; 5,510,267 ; 5,516,695 ; 5,648,225 and EP-A-161770 .
  • Several such agents are available commercially, including FACSTM Lysing Solution (Becton Dickinson Immunocytometry Systems, San Jose, Calif., catalogue No. 349202) and Whole Blood Lysing Solution (Caltag Laboratories, Inc., Burlingame, Calif., catalogue no. GAS-10). The minimum duration of incubation with lysis reagent depends upon whether, after lysis, debris may be removed by centrifugation.
  • Optical labels used with the invention may include direct or indirect attachment of fluorescent moieties, colorimetric moieties and chemiluminescent moieties, to binding compounds.
  • Reviews of labeling methodology that provide guidance for selection and attachment of labels to binding compounds include Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002 ); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993 ) and Hermanson, Bioconjugate Techniques (Academic Press, New York, 1996 ).
  • Particular dyes applicable to the invention are disclosed in the following sample of references: Menchen et al., U.S.
  • patent 5,188,934 (4,7-dichlorofluorscein dyes); Begot et al., U.S. patent 5,366,860 (spectrally resolvable rhodamine dyes); Lee et al., U.S. patent 5, 847,162 (4,7-dichlororhodamine dyes); Khanna et al., U.S. patent 4,318,846 (ether-substituted fluorescein dyes); Lee et al., U.S. patent 5,800,996 (energy transfer dyes); Lee et al., U.S. patent 5,066,580 (xanthene dyes) and Mathies et al. U.S.
  • patent 5,688,648 energy transfer dyes
  • Labeling can also be carried out with quantum dots, as disclosed in the following patents and patent publications: 6,322,901 ; 6,576,291 ; 6,423,551 ; 6,251,303 ; 6,319,426 ; 6,426,513 ; 6,444,143 ; 5,990,479 ; 6,207,392 ; 2002/0045045 and 2003/0017264 .
  • fluorescent signal generating moiety means a signaling means which conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence life time, emission spectrum characteristics and energy transfer.
  • optical labels of the invention are fluorescent signal generating moieties.
  • fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR
  • Fluorescence resonsant energy transfer (FRET) tandem fluorophores may also be used, such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7; also, PE-Alexa dyes (610, 647, 680) and APC-Alexa dyes.
  • PerCP is described in U.S. patent 4,876,190 .
  • Cyanine resonance energy transfer tandem fluorophores (“tandem fluorophores", “tandem dyes", “tricolor stains”) have recently expanded the choices of fluorophore available for single-laser, multi-color flow cytometric analysis.
  • PE-CY5 tandem staining proves particularly well-suited for three-color analysis: the R-PE moiety, excited by the 488 nm light of an argon ion laser, serves as an energy donor, and CY5, acting as an energy acceptor, fluoresces at 670 nm, readily distinguishable from the emission of FITC and PE. Cyanine fluorophores are described in U.S. Pat Nos. 5,268,486 ; 4,337,063 ; 4,404,289 ; 4,405,711 ; and in Mujumdar et al., Bioconj. Chem.
  • optical signals may be measured by a variety of conventional instruments, particularly microscopes and flow cytometers.
  • the determination of absolute or relative numbers of lymphocytes or a subset of lymphocytes in a sample may be accomplished by identifying a subset of data that forms a distinguishable cluster within the totality of data. Identification of distinct clusters in either univariate or multi-variate data is well-known to those of ordinary skill, particularly for data acquired from measurements using a flow cytometer, as exemplified by the following references: Bagwell et al., J. Histochem. Cytochem., 27: 293-296 (1979 ); Young, J. Histochem.
  • enumeration of cell types using flow cytometers or automated microscopes includes setting gating parameters, e.g. upper and lower signal values of the one or more optical labels used, which are characteristic of the cell types of interest. The instrument then automatically tabulates cell types corresponding to the selected parameters.
  • Exemplary microscopes for slide-based analysis of samples include an iCyteTM Automated Imaging Cytometer (CompuCyte Corp., Cambridge, MA)(e.g. Kamensky et al., Cytometry, 12A: 381 (1991 )); an Axioplan 2 MOT microscope (Carl Zeiss, Goettingen, Germany), e.g. equipped with 100W Mercury lamp, 12 bit Axiocam CCD camera, and motorized object desk and filter changer.
  • iCyteTM Automated Imaging Cytometer CompuCyte Corp., Cambridge, MA
  • Axioplan 2 MOT microscope Carl Zeiss, Goettingen, Germany
  • kits for detection of specific cell types using labeled binding compounds will be particularly useful.
  • a test kit typically comprises one or more reagents, such as one or more labeled binding compounds, packaged in a container, such as, without limitation, a vial, tube or bottle, in a package suitable for commercial distribution, such as, without limitation, a box, a sealed pouch, a blister pack and a carton.
  • kits are disclosed for identifying and counting lymphocytes in whole blood, which kits comprise a first binding compound specific for CD45RA and a second binding compound specific for a cell surface marker common to T-lymphocytes, wherein the first and second binding compounds have the same label.
  • the cell surface marker common to T lymphocytes is a T lymphocyte-specific marker, such as CD2 or CD3.
  • the first and second binding compounds are labeled with a fluorescent dye, such as a fluorescent dye selected from those listed above.
  • binding compounds are preferably labeled with a fluorescent dye selected from the group consisting of phycoerythrin (PE), peridinin chlorophyll protein-Cy5.5 (PerCP-Cy5.5), PE-Cy7, allophycocyanin (APC), Alexa Fluor 647, Cy5, APC-Cy5.5, APC-Cy7, or APC-Alexa Fluor 750.
  • such group consists of phycoerythrin (PE), peridinin chlorophyll protein-Cy5.5 (PerCP-Cy5.5), PE-Cy7, allophycocyanin (APC), APC-Cy5.5, or APC-Cy7.
  • PE phycoerythrin
  • PerCP-Cy5.5 peridinin chlorophyll protein-Cy5.5
  • PE-Cy7 allophycocyanin
  • APC-Cy5.5 APC-Cy5.5
  • APC-Cy7 APC-Cy7
  • Kits are disclosed for determining the percentage of one or more types within a sample of white blood cells, for example, contained within a sample of whole blood.
  • such kits comprise (i) a first and second binding compound as described above, (ii) a third binding compound specific for a monocyte-specific marker, such as CD 14, wherein a portion of such third binding compound is labeled with the same optical label as the first and second binding compound (a first optical label) and the remainder being labeled by a distinct optical label (a second optical label), and (iii) a fourth binding compound specific for a granulocyte-specific marker, such as CD15, wherein such fourth binding compound is labeled with the second optical label.
  • the third binding compounds are labeled with an optical label (a third optical label) distinct from the first and second optical labels.
  • an optical label a third optical label
  • the first, second, and/or third optical labels are fluorescent labels.
  • a first optical label is PerCP and a second optical label is APC.
  • a first optical label is PerCP
  • a second optical label is APC
  • a third optical label is PE-Cy5.5.
  • such optical labels may be interchanged among the respective binding compounds for additional forms of the compositions and kits.
  • kits may be packaged together or separately, and each constituent may be presented in one or more tubes or vials, or in cartridge form, as is appropriate.
  • the constituents, independently or together, may be packaged in any useful state, including without limitation, in a dehydrated, lyophilized, classified or aqueous state.
  • the kits may be in the form of a disposable cartridge having binding compounds incorporated in appropriate reaction chambers as dried reagents. In particular, such dried reagents may be incorporated as films and/or coatings within such cartridge.
  • labeled binding compounds were prepared and tested on whole blood samples from normal individuals.
  • Commercially available reagents e.g. BD Biosciences, San Jose, CA
  • scatter and fluorescent intensity measurements were made on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA).
  • Fig. 1A Box (106) indicates a cluster corresponding to lymphocytes.
  • Fig. 1B displays data from lymphocytes in counts versus PE fluorescence histogram in which total lymphocytes (100) subpopulations of lymphocytes (102 for CD16+ or CD56+, and 104 for CD20+) are indicated.
  • Low PE-fluorescence high scatter cells (108) correspond to non-lymphocyte white blood components, such as granulocytes and monocytes.
  • Fig. 1C is a table giving count data corresponding to Figs. 1A and 1B .
  • Lymphocytes correspond to high PE fluorescence in region (200) that is distinguishable from cells with low PE fluorescence values (e.g. clusters 206 and 208).
  • lymphocytes (200) CD4+ lymphocytes are shown in high-PE high-APC fluorescence cluster (202), whereas non-CD4+ lymphocytes are shown in a clearly distinguishable high-PE low-APC fluorescence cluster (204).
  • Separate clusters of high APC low PE fluorescence (206) and low APC low PE fluorescence (208) correspond to monocytes and granulocytes, respectively.
  • Fig. 3A where data cluster in region (300) corresponds to CD4+ lymphocytes and data cluster in region (302) corresponds to non-CD4+ lymphocytes.
  • Fig. 3B is a tabulation of counts of the indicated cell types based on the imaging measurements and counts using a commercially available staining assay (TriTest, BD Biosciences, San Jose, CA) with fluorescence measurements using a flow cytometer.
  • the binding compound was an anti-CD45RA antibody labeled with FITC and components of a commercially available kit (TriTest CD3/4/45) that contains anti-CD3, anti-CD4, and anti-CD45 antibodies labeled with PE.
  • 50 ⁇ L whole blood was combined with 20 ⁇ L staining cocktail (TriTest alone) or 25 ⁇ L staining cocktail (TriTest reagent plus CD45RA stain), and incubated for 15 minutes at room temperature in the dark, after which 450 ⁇ L of red blood cell lysing agent (FACSLyse, BD Biosciences, San Jose, CA) was added.
  • FACSLyse red blood cell lysing agent
  • Antibody or "immunoglobulin” means a protein, either natural or synthetically produced by recombinant or chemical means, that is capable of specifically binding to a particular antigen or antigenic determinant.
  • Antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V L ) and a. constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • the constant domains are not involved directly in binding an antibody to an antigen.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG,, IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
  • immunoglobulins e.g., IgG, IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
  • Antibody fragment and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e.
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2 , and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or "single chain polypeptide"), including (1)single-chain Fv (scFv) molecules; (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety; and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments.
  • mAb monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each mAb is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
  • Antigenic determinant means a site on the surface of a molecule, usually a protein, to which a single antibody molecule binds; generally a protein has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • a preferred antigenic determinant is a phosphorylation site of a protein.
  • Binding compound means a compound that is capable of specifically binding to a particular target molecule.
  • binding compounds include antibodies, lectins, nucleic acids and aptamers, e.g. Sharon and Lis, Lectins, 2nd Edition (Springer, 2006 ); Klussmann, The Aptamer Handbook: Functional Oligonucleotides and Their Applications (John Wiley & Sons, New York, 2006 ).
  • binding compounds are antibodies, and more preferably, binding compounds are monoclonal antibodies.
  • CD2 means a cell surface molecule of the cluster designation (CD) system, also known as T11 or LFA-2, that is expressed on T cells, thymocytes, and NK cells, and that functions as an adhesion molecule, binding CD58 (LFA-3).
  • CD2 refers to the human CD2 molecule.
  • CD3 means a cell surface molecule of the cluster designation (CD) system, also known as T3, that is expressed on T cells and thymocytes, and that is associated with the T cell antigen receptor (TCR), is required for cell surface expression of and signal transduction by TCR. Its cytoplasmic domains contain ITAM motifs and bind cytoplasmic tyrosine kinases. Preferably, CD3 refers to the human CD3 molecule.
  • CD4 means a cell surface molecule of the cluster designation (CD) system, also known as T4 or L3T4, that is expressed on thymocyte subsets, helper and inflammatory T cells (about two thirds of peripheral T cells), monocytes, macrophages, and that functions as coreceptor for MHC class II molecules, binds Lck on cytoplasmic face of membrane.
  • CD4 is a receptor for HIV-I and HIV-2 gp120.
  • CD4 refers to the human CD4 molecule.
  • CD45RA means a cell surface molecule of the cluster designation (CD) system that is expressed on B cells, T cell subsets (naive T cells), and monocytes, and that is a splice variant of CD45 containing the A exon.
  • CD45RA refers to the human CD45RA molecule.
  • CD56 means a cell surface molecule of the cluster designation (CD) system, also known as NKH-1, that is expressed on NK cells, and that is a splice variant of the Neural Cell Adhesion Molecule (NCAM).
  • CD56 refers to the human CD56 molecule.
  • “Complex” as used herein means an assemblage or aggregate of molecules in direct or indirect contact with one another.
  • "contact,” or more particularly, “direct contact” in reference to a complex of molecules, or in reference to specificity or specific binding means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions dominate the interaction of the molecules.
  • a complex of molecules is stable in that under assay conditions the complex is thermodynamically more favorable than a non-aggregated, or non-complexed, state of its component molecules.
  • “complex” usually refers to a stable aggregate of two or more proteins.
  • a “complex” refers to a stable aggregate of two proteins, such as an antibody specifically bound to an antigenic determinant of a target protein.
  • “Dried reagents” mean assay reagents, such as buffers, salts, active compounds, such as enzymes and cofactors, or binding compounds, such as antibodies or aptamers that are provided in a dehydrated formulation for the purpose of improved shelf-life, ease of transport and handling and improved storage.
  • the nature, composition, and method of producing dried reagents vary widely and the formulation and production of such materials is well-known to those of ordinary skill in the art as evidenced by the following references: Franks et al., U.S. patent 5,098,893 ; Cole, U.S. patent 5,102,788 ; Shen et al., U.S. patent 5,556,771 ; Treml et al., U.S.
  • Dried reagents include solid and/or semi-solid particulates, powders, tablets, crystals, films and coatings, that are manufactured in a variety of ways.
  • dried reagents are lyophilized coatings or films on the inner walls of vessels or inside a chamber of a disposable cartridge for carrying out an assay in accordance with the invention.
  • Dried reagents may include excipients, which are usually inert substances added to a material in order to confer a suitable consistency or form to the material.
  • excipients are usually inert substances added to a material in order to confer a suitable consistency or form to the material.
  • excipients are known to those of skill in the art and can comprise a number of different chemical structures.
  • excipients examples include carbohydrates, such as sucrose, glucose, trehalose, melezitose, dextran, and mannitol; proteins such as BSA, gelatin, and collagen; and polymers such as PEG and polyvinyl pyrrolidone (PVP).
  • the total amount of excipient in the lyophilized particulate may comprise either single or multiple compounds.
  • the type of excipient is a factor in controlling the amount of hygroscopy of a dried reagent. Lowering hygroscopy can enhance the a dried reagent's integrity and cryoprotectant abilities. However, removing all water from such a composition would have deleterious effects on those reaction components, proteins for example, that require certain amounts of bound water in order to maintain proper conformations.
  • Gram-specific marker means any molecule that is present on or in substantially all granulocytes, but is substantially absent from other white blood cell types.
  • Exemplary granulocytes-specific markers include, the following molecules: 1C3, 3C4, 6D10, 2B2, 8C5, alkaline phosphatase, calprotectin, CD18, CD15, CD16, CD24, CD32, CD34, CD45, CD66b, CEACAM8, DH59B, EMR3, eosinophil cationic protein, granulocyte factor, GMP, Gr-1 (Ly-G6), granulocyte elastase, HIS48, IL-8, leukocyte alkaline phosphatase, LRG, myeloperoxidase and NKH1.
  • granulocytes-specific markers are cell surface molecules. More preferably, granulocytes-specific markers are the human equivalent of the markers listed above. Still more preferably, a granulocytes-specific marker is CD15.
  • Kit means to any delivery system for delivering materials or reagents for carrying out a method of the invention.
  • delivery systems include systems that allow for the storage, transport, or delivery of assay reagents (e.g., probes and ancillary labeling antibodies in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay) from one location to another.
  • assay reagents e.g., probes and ancillary labeling antibodies in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. Such contents may be delivered to the intended recipient together or separately.
  • a first container may contain buffers and a reaction cuvette orvessel for use in an assay, while a second container may contain probes.
  • Optional additional components may include, sample-extraction apparatus, e.g. skin puncturing devices for exposing small amounts of blood or germicidal swabs.
  • Monocyte-specific marker means any molecule that is present on or in substantially all monocytes, but is substantially absent from other white blood cell types.
  • Exemplary monocyte-specific markers include the following molecules: 125I-WVH-1, 63D3, CB12, CD11a, CD14, CD15, CD54, CD62L, CD163, cytidine deaminase, DH59B, Fc-receptors, Flt-1, hMGL, Ki-M1p, Leu-7, lysozyme, leucocyte tartrate-resistant acid phosphatase, mannosyl receptors, peanut agglutinin, thromboplastin, thymidine phosphorylase, TNF and urokinase.
  • monocytes-specific markers are cell surface molecules. More preferably, monocytes-specific markers are the human equivalent of the markers listed above. Still more preferably, a monocytes-specific marker is CD 14.
  • sample means a quantity of material from blood sample in which detection or measurement of target cells, particles, beads, and/or analytes is sought.
  • a blood sample may include a specimen of synthetic origin.
  • Blood samples may be from animal, including human and may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including such animals as ungulates, bear, fish and rodents. These examples are not to be construed as limiting the sample types applicable to the method of the present invention.
  • sample and “specimen” are used interchangeably.
  • “Specific” or “specificity” in reference to the binding of one molecule to another molecule means the recognition, contact, and formation of a stable complex between the two molecules, together with substantially less recognition, contact, or complex formation of that molecule with other molecules.
  • “specific” in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with another molecules in a reaction or sample, it forms the largest number of the complexes with the second molecule. Preferably, this largest number is at least thirty percent.
  • molecules involved in a specific binding event have areas on their surfaces, and/or in the case of proteins in cavities, giving rise to specific recognition between the molecules binding to each other.
  • specific binding examples include antibody-antigen interactions, enzyme-substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides and receptor-ligand interactions.
  • contact in reference to specificity or specific binding means two molecules are close enough that weak noncovalent chemical interactions, such as Van der Waal forces, hydrogen bonding, base-stacking interactions, ionic and hydrophobic interactions dominate the interaction of the molecules.
  • “Spectrally resolvable" in reference to a plurality of fluorescent labels, or dyes means that the fluorescent emission bands of the dyes are sufficiently distinct, e.g. non-overlapping, that binding compounds to which the respective dyes are attached can be distinguished on the basis of the fluorescent signal generated by the respective dyes by conventional photodection systems, e.g. employing a standard system of filters, mirrors, dichoics, photomultiplier tubes or photodiodes such as described in the following references: Wheeless et al., Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985 ); Shapiro (cited above).
  • T lymphocyte-specific marker means any molecule that is present on or in substantially all T lymphocytes, but is substantially absent from other white blood cell types.
  • Exemplary T lymphocyte-specific markers include the following molecules: CD1a, CD1d, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD45RO, CD72, CD134, CD150, CRTAM, FOXP3, FT2, GPCA, HML-1, HT23A, Leu-22, Ly-2, Ly-m22, MICG, MRC OX-8, MRC OX-22, OX40, PD-1, RT6, TCR, Thy-1 (CD90) and TSA-2.
  • T lymphocyte-specific markers are cell surface molecules. More preferably, T lymphocyte-specific markers are the human equivalent of the markers listed above. Still more preferably, T lymphocyte-specific markers are CD2 or CD3 molecules.

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Claims (6)

  1. Procédé de numération en nombre absolu des lymphocytes présents dans un échantillon sanguin, le procédé comprenant les étapes suivantes :
    - combiner ledit échantillon avec un premier composé de liaison marqué par un colorant fluorescent et spécifique d'un marqueur spécifique des lymphocytes T qui est CD2 ou CD3, et un second composé de liaison marqué par un colorant fluorescent et spécifique de CD45RA, dans un mélange, dans lequel ledit premier composé de liaison et ledit second composé de liaison sont marqués à l'aide d'un même colorant fluorescent ;
    - mesurer les signaux de fluorescence desdits composés de liaison marqués au colorant fluorescent qui se sont liés à des cellules dans ledit échantillon ; et
    - en l'absence de mesure de diffusion latérale, compter les lymphocytes dans ledit échantillon.
  2. Procédé selon la revendication 1, dans lequel lesdits premier et second composés de liaison sont des anticorps.
  3. Procédé de détermination en pourcentage des lymphocytes T auxiliaires présents dans un échantillon sanguin, ledit procédé comprenant les étapes suivantes :
    - combiner ledit échantillon avec un premier composé de liaison marqué par un colorant fluorescent et spécifique d'un marqueur spécifique des lymphocytes T qui est CD2 ou CD3, et un second composé de liaison marqué par un colorant fluorescent et spécifique de CD45RA, dans un mélange, dans lequel ledit premier composé de liaison et ledit second composé de liaison sont marqués à l'aide d'un premier colorant fluorescent, et un troisième composé de liaison marqué par un colorant fluorescent et spécifique de CD4, dans lequel ledit troisième composé de liaison est marqué par un colorant fluorescent différent dudit premier colorant fluorescent ;
    - mesurer les signaux de fluorescence desdits composés de liaison marqués au colorant fluorescent qui se sont liés à des cellules dans ledit échantillon ; compter les lymphocytes dans ledit échantillon ;
    - en l'absence de mesure de diffusion latérale, compter les lymphocytes T auxiliaires dans ledit échantillon ; et
    - déterminer le pourcentage des lymphocytes T auxiliaires présents dans ledit échantillon.
  4. Procédé selon la revendication 3, dans lequel lesdits premier, second et troisième composés de liaison sont des anticorps.
  5. Utilisation d'un kit comprenant un premier composé de liaison marqué par un colorant fluorescent et spécifique d'un marqueur spécifique des lymphocytes T qui est CD2 ou CD3, et un second composé de liaison marqué par un colorant fluorescent et spécifique de CD45RA, dans laquelle ledit premier composé de liaison et ledit second composé de liaison sont marqués à l'aide d'un même colorant fluorescent, pour effectuer la numération en nombre absolu des lymphocytes présents dans un échantillon sanguin à l'aide du procédé selon la revendication 1.
  6. Utilisation selon la revendication 5, dans laquelle lesdits premier et second composés de liaison sont des anticorps.
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